This invention relates to methods and compositions for the treatment of conditions associated with vascular insufficiency, and to methods and compositions for screening assays to select agents that are useful for this purpose. In particular, the invention relates to HMG CoA reductase inhibitors and their use in promoting angiogenesis in vivo and in activating Akt in vascular endothelial cells in vitro and in vivo.
Akt (c-Akt) is a proto-oncogene encoding a serine-threonine kinase (Testa, J. R. and Bellacosa, A., Leukemia Res., 1997, 21:1027-1031). It is the cellular homolog of the viral oncoprotein v-Akt, and is related to protein kinase-C (PKC) within the catalytic domain. However, c-Akt differs from the PKC family members by the presence of a pleckstrin homology (PH) domain at its N-terminus that is involved in the regulation of the activity of the enzyme by growth factors and intracellular signaling molecules. Various extracellular stimuli reportedly activate Akt through the phosphoinositide 3-kinase (PI 3-kinase) pathway (Datta, K. et al., J. Biol. Chem., 1996, 271:30835-30839; Franke, T. F., et al., Cell, 1995, 81:727-736; King, W. G. et al., Mol. Cell. Biol., 1997, 17:4406-4418). The lipid products of the PI 3-kinase reaction reportedly may activate Akt either by binding to the Akt pleckstrin homology (PH) domain (Franke, T. F. et al., 1997, Cell, 88:435:437), or by activating a protein kinase that phosphorylates Akt (Kohn, A. D., et al., J. Biol. Chem., 1996, 271:21920-21926; Stokoe et al., Science, 1997, 277:567-570). Activation of Akt reportedly inhibits apoptosis induced by growth factor withdrawal or irradiation in neural cells, fibroblasts, and lymphocytes (Franke, T. F., et al., Science, 1997, 275:665-668; Hemmings, Science, 1997, 275:628-630). Recently, it has been reported that Akt phosphorylates the pro-apoptotic protein Bad leading to Bad inactivation and cell survival (Datta, K., et al., Cell, 1997, 91:231-241; Peso, L., et al., Science, 1997, 278:687-689).
Despite the foregoing speculation, the precise mechanism underlying Akt activation in vivo has yet to be elucidated. Accordingly, a need still exists to identify the precise mechanism underlying Akt activation in vivo and to use such knowledge to develop methods and compositions for treating conditions that are amenable to treatment by Akt activation in vivo, as well as to develop screening assays that are useful for identifying agents which activate Akt in vivo and in vitro.
HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is the enzyme which catalyzes the rate limiting step of cholesterol biosynthesis. HMG-CoA reductase inhibitors, also known as statins, are molecules which inhibit the enzymatic activity of HMG-CoA reductase and have been used to treat patients suffering from hypercholesterolemia. The first such inhibitor (compactin or Mevastatin) was isolated in 1976 (Endo, A. et al., F.E. B.S. Lett., 72: 323-326, 1976) and since then many other natural and chemically modified versions of Mevastatin have been identified and developed for clinical use, including Lovastatin, and Simvastatin.
Recent studies have shown that, in addition to treatment of hyperlipidemia, HMG-CoA reductase inhibitors are useful in the treatment of acne and/or skin aging (see, e.g. Breton, L. et al., U.S. Pat. No. 5,902,805); can increase nitric oxide (NO)-mediated vasodilation and blood vessel relaxation (see e.g., Liao, J. K. et al., WO 99/18952); and can help prevent a second or additional myocardial infarction (see, e.g., Behounek, B. D. et al., U.S. Pat. No. 5,674,893; Olukotun, A. Y. et al., U.S. Pat. No. 5,622,985).
Additonally, several studies have shown that HMG-CoA reductase inhibitors have anti-angiogenic activity. Feleszko, W. et al., Int. J. Cancer, 81: 560-567 (1999) reported that treatment of a mouse model of tumor-cell induced angiogenesis with a combination of TNF-xcex1 and lovastatin produced a significant inhibition of tumor-induced blood-vessel formation whereas treatment with either TNF-xcex1 or lovastatin alone showed no angiostatic effects. Jones, M. K. et al., Am. J. Physiol., 276: G1345-GI1355 (1999) reported that mevastatin, an inhibitor of Ras activation, completely blocked the induction of VEGF (a potent angiogenic factor) expression in cultured primary endothelial cells. Kong, D. et al., Circulation, 100(18): I-39, Abstract #194 (1999) reported that simvastatin exerted potent anti-angiogenic effects independent of its cholesterol lowering effects.
To understand the mechanism by which angiogenic agents such as VEGF promote new blood vessel formation (angiogenesis), we have analyzed the signaling pathways downstream from the growth factor. In particular, we have analyzed Akt signaling in endothelial cells. Akt (also known as Protein Kinase-B, PKB) inhibits apoptotic cell-death and, in particular, inhibits apoptotic cell-death of cardiomyocytes, skeletal myocytes and/or vascular endothelial cells. (See, e.g., U.S. Ser. No. 9/922,633, entitled, xe2x80x9cAkt Compositions for Enhancing Survival of Cellsxe2x80x9d, PCT Application No. PCT/US99/22633, published as WO 00/20025). In the course of analyzing Akt signaling events in an animal model of ischemia, we have surprisingly discovered that activation of Akt signaling in endothelial cells is sufficient to promote angiogenesis. We also have discovered that HMG CoA reductase inhibitors such as simvastatin are potent activators of Akt. In view of these discoveries, we believe that simvastatin and other HMG CoA reductase inhibitors can be used to promote angiogenesis in tissues, and that such inhibitors are useful for treating conditions in which new blood vessel growth is desirable to treat the condition. These discoveries were highly unexpected in view of previous research indicating that HMG CoA reductase inhibitors had angiostatic activity.
According to one aspect of the invention, a method for promoting angiogenesis in a tissue of a subject in need of such treatment is provided. The method involves administering to the subject, an HMG CoA reductase inhibitor in an amount effective to promote angiogenesis in the tissue, preferably in situations where the subject is not otherwise in need of administration of an HMG CoA reductase inhibitor. According to this aspect, the subject may or may not be hyperlipidemic and/or hypercholesterolemic. In certain embodiments, the method further includes the step of detecting angiogenesis in the tissue.
Conditions that can be treated in accordance with this method of the invention (administration by any route, preferably oral administration) are conditions characterized by insufficient vascularization (or predisposition thereto) of the affected tissue, i.e., conditions in which neovascularization (rather than increases in nitric oxide (NO)-mediated vasodilation) is needed to achieve sufficient vascularization in the affected tissue, and that are selected from the following group of conditions: (1) diabetic ulcers, (2) gangrene, (3) surgical or other wounds requiring ncovascularization to facilitate healing; (4) Buerger""s syndrome; (5) hypertension; (6) ischemic diseases including, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, myocardial ischemia, ischemia of tissues such as, for example, muscle, brain, kidney and lung; and other conditions characterized by a reduction in microvasculature. The preferred method of treatment further includes the step of detecting angiogenesis in the affected tissue following treatment. Exemplary tissues in which angiogenesis can be promoted and, optionally, detected in accordance with this method of the invention include: hypertension; ulcers (e.g., diabetic ulcers); surgical wounds; ischemic tissue, i.e., a tissue having a deficiency in blood as the result of an ischemic disease including, for example, muscle, brain, kidney and lung; ischemic diseases including, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy and myocardial ischemia. In the preferred embodiments, the HMG CoA reductase inhibitor is a statin molecule (see description below). More preferably, the statin molecule(s) is orally administered.
According to yet another aspect of the invention, a method for promoting angiogenesis in a tissue of a subject is provided. The method involves locally administering to the tissue, an HMG CoA reductase inhibitor in an amount effective to promote angiogenesis in the tissue. Preferably, the subject is not otherwise in need of administration (particularly, local administration) of an HMG CoA reductase inhibitor. According to certain embodiments, the subject is hyperlipidemic and/or hypercholesterolemic. According to yet other embodiments, the subject is nonhyperlipidemic and/or nonhypercholesterolemic.
Conditions that can be treated in accordance with this method of the invention (locally administering the therapeutic agent to the tissue) are conditions characterized by insufficient vascularization of the affected tissue, i.e., conditions in which neovascularization, rather than nitric oxide mediated vasodilation, is desirable to achieve sufficient vascularization in the affected tissue. Exemplary conditions that can be treated in accordance with the methods of the invention include: (1) severe occlusive and/or obstructive vascular disease, such as (a) peripheral vascular disease (particularly, diabetic peripheral vascular disease), (b) myocardial ischemia /myocardial infarction, (c) coronary artery disease, (d) cerebral vascular disease, (e) visceral vascular disease; and (2) surgical or other wounds requiring neovascularization to facilitate healing. The preferred method of treatment further includes the step of detecting angiogenesis in the affected tissue following treatment. Exemplary tissues to which the HMG CoA reductase inhibitor can be administered in accordance with the methods of the invention to promote angiogenesis therein include cardiac tissue, ulcers (e.g., diabetic ulcers), surgical wounds, neuronal tissue (e.g., tissue damaged incident to ischemia of the brain), and other tissue damaged as a result of severe occlusive and/or obstructive vascular disease or injury.
An HMG CoA reductase inhibitor is a term of art which refers to a molecule which inhibits the enzymatic activity of the enzyme, HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase. As used herein, the HMG CoA reductase inhibitors that are useful in accordance with the methods of the invention satisfy the conventional meaning of this phrase and are capable of activating Akt signaling in vascular endothelial cells. As used herein, xe2x80x9cactivating Akt signalingxe2x80x9d refers to inducing a change in the Akt polypeptide that is sufficient to promote angiogenesis when the activation occurs in vivo. In general, the change that is induced is phosphorylation of the Akt polypeptide, typically at Ser 473 and/or Thr 308. Exemplary HMG CoA reductase inhibitors are provided in the detailed description of this invention and in the references and/or patent documents identified therein. The preferred HMG CoA reductase inhibitors that are useful in accordance with the methods and compositions of the invention are statin molecules. These include: Lovastatin (Mevacor), Pravastatin (Pravachol), Simvastatin (Zocor), Fluvastatin (Lescol), Atorvastatin (Lipitor), or Cerivastatin (Baycol), provided that when the statin molecule is an inhibitor of HMG CoA, it is processed into the corresponding lactone form prior to local administration. Also provided are screening assays for selecting novel inhibitors which are capable of activating Akt signaling in vascular endothelial cells.
The preferred methods of treatment for this aspect of the invention involve locally administering the HMG CoA reductase to the tissue of a subject in an amount effective to promote angiogenesis in the tissue. In certain embodiments, locally administering involves inserting a stent containing the HMG CoA reductase inhibitor into the tissue. Alternatively, locally administering involves administering to the subject a pharmaceutical composition containing an HMG CoA reductase inhibitor and a pharmaceutically acceptable carrier. In certain preferred embodiments, the pharmaceutical composition is suitable for topical application or internal applications and can be formulated as a salve, a gel or a patch. Preferably, the pharmaceutical composition is a controlled release matrix and, more preferably, the composition is formulated to release the HMG CoA reductase inhibitor substantially continuously for a period of at least one day (i.e. a sustained release formulation).
According to yet another aspect of the invention, a method for activating an Akt polypeptide is provided. The method involves contacting a cell containing an Akt polypeptide with an HMG CoA reductase inhibitor in vitro under conditions wherein the HMG CoA reductase inhibitor (preferably a statin molecule as described herein) activates the Akt polypeptide. By xe2x80x9cactivatexe2x80x9d it is meant that the HMG CoA reductase inhibitor facilitates the transformation of the Akt polypeptide from an inactive to an active form. This transition can be determined by detecting various parameters, e.g., degree of Akt polypeptide phosphorylation, degree of phosphorylation of an Akt substrate molecule (e.g. Bad, histone H2B, eNOS, etc., or fragments thereof), or other downstream signaling events, including for example a change in the rate of protein degradation, a change in the level of mRNA transcription, a change in the level of protein translation, reduction of apoptosis, induction of angiogenesis, etc. Although not wishing to be bound to any particular theory or mechanism, it is believed that HMG CoA reductase inhibitors facilitate the phosphorylation of the Akt polypeptide (e.g., at Ser 473 and/or Thr 308) which mediates further signaling events that result in angiogenesis in vivo. Thus, in particularly preferred embodiments such as those methods useful as screening assays, the Akt polypeptide that is useful in this aspect of the invention is an Akt polypeptide that is expressed by a vascular endothelial cell. Exemplary Akt polypeptides are described in U.S. Ser. No. 9/922,633, entitled, xe2x80x9cAkt Compositions for Enhancing Survival of Cellsxe2x80x9d, PCT Application No. PCT/US99/22633, published as WO 00/20025. The preferred Akt polypeptide has SEQ ID NO. 1. Exemplary conditions for performing this aspect of the invention are provided in the Examples.
According to still another aspect of the invention, a method for promoting angiogenesis by activating an Akt molecule is provided. The method involves contacting a cell containing an Akt polypeptide with an HMG CoA reductase inhibitor in vitro under conditions wherein the HMG CoA reductase inhibitor (preferably a statin molecule as described herein) activates the Akt polypeptide. While not wishing to be bound by theory, it is believed that activation of Akt allows Akt molecules to initiate signalling events which lead to promotion of angiogenesis. In particularly preferred embodiments such as those methods useful as screening assays, the Akt polypeptide that is useful in this aspect of the invention is an Akt polypeptide that is expressed by a vascular endothelial cell. Exemplary Akt polypeptides are described in U.S. Ser. No. 9/922,633, entitled, xe2x80x9cAkt Compositions for Enhancing Survival of Cellsxe2x80x9d, PCT Application No. PCT/US99/22633, published as WO 00/20025. The preferred Akt polypeptide has SEQ ID NO. 1.
According to yet another aspect of the invention, a screening method to identify putative HMG CoA reductase inhibitors that activate an Akt polypeptide is provided. The method involves performing an Akt polypeptide activation (e.g., phosphorylation) assay in the presence and absence of a putative HMG CoA reductase inhibitor; and determining the level of Akt polypeptide activation in the presence and absence of the putative inhibitor, wherein an increase in the level of Akt polypeptide activation in the presence of the putative inhibitor relative to the level of Akt polypeptide activation in the absence of the putative inhibitor indicates that the putative inhibitor is an HMG CoA reductase inhibitor as used herein.
According to still another aspect of the invention, a method for treating a wound (e.g., a surgical wound) is provided. The method involves contacting the wound with a sufficient amount of an HMG CoA reductase inhibitor under conditions wherein the HMG CoA reductase inhibitor causes neovasculatization and enhances healing of the wound. In certain embodiments, contacting the wound involves locally administering the HMG CoA reductase inhibitor to the wound. For example, the HMG CoA reductase inhibitor can be contained in a pharmaceutical composition that is formulated for local administration to a wound or to a tissue in need of neovascularization in a subject.
According to a further aspect of the invention, a pharmaceutical composition is provided. The composition comprises an HMG CoA reductase inhibitor (preferably, a statin molecule as described herein); and a pharmaceutically acceptable carrier suitable for local delivery to a wound or a tissue in need of neovascularization in a subject. In certain embodiments, the composition is suitable for non-oral, preferably topical or intramuscular applications. In these and other embodiments, the composition optionally is formulated as a salve, a gel, or a patch. In particularly preferred embodiments, the composition is a controlled release matrix and, more preferably, the composition is formulated to release the HMG CoA reductase inhibitor substantially continuously for a period of at least a day. Methods for preparing such pharmaceutical compositions are also provided.
According to yet another aspect of the invention, the pharmaceutical composition may additionally comprises an angiogenic growth factor. Preferred angiogenic growth factors include, for example, acidic and basic fibroblast growth factors, vascular endothelial growth factor, epidermal growth factor, transforming growth factor xcex1 and xcex2, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor a, hepatocyte growth factor, insulin like growth factor, etc. In a particular embodiment the pharmaceutical composition contains an Akt protein, preferably in a constitutively active form. According to still another aspect of the invention, the pharmaceutical composition may comprise a nucleic acid encoding for an angiogenic growth factor or an Akt protein.
In a particularly preferred embodiment, coadministration of an HMG CoA reductase inhibitor with an Akt molecule produces a syngeristic effect wherein the resultant angiogenesis observed with the coadministration is greater than the angiogenesis that would be expected from the additive effects when either the HMG CoA reductase inhibitor or the Akt molecule is administered alone.
Pharmaceutical compositions comprising angiogenic growth factor proteins, an Akt protein or a nucleic acid encoding for an angiogenic growth factor or an Akt protein, are preferably administered locally to a site requiring angiogenesis via direct injection or intraarterially via catheter delivery. By xe2x80x98direct injectionxe2x80x99 it is meant that a syringe or needle is used to intramuscularly or subcutaneously inject the pharmaceutical composition to the desired site using standard injection techniques. By xe2x80x98catheter deliveryxe2x80x99 it is meant that the pharmaceutical composition is delivered intraarterially using any type of balloon catheter well know in the art, including, for example, double balloon catheters, porous balloon catheters and hydrogel coated balloon catheters (see e.g. Riessen, R. et al., J. Am. Coll. Cardiol., 23(5): 1234-1244, 1994).
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments.